The Structural Mass Spectrometry Group now concentrates almost entirely on peptide and protein type samples. These may come to the laboratory as spots on gels, recombinant protein samples for structural confirmation or post-translational modification determination, as proteins or protein complexes precipitated or purified with antibodies, or as entire proteomes. Spots on gels are routinely identified by digestion of the spot with trypsin, after reduction and alkylation, and subsequent analysis on the QTOF mass spectrometer in the group. These MS/MS results (including sequence data from the peptides) can be searched on in-house software or against the new NIH Mascot server. Our group has specialized in identifying the post-translational modifications in proteins. In particular, we have examined many proteins and characterized their disulfides. We also are working hard to develop methods for the characterization of glycosylation sites. N-linked glycoforms are easily removed with PGNaseF, and the Asn is changed to an Asp thereby leaving a marker. O-linked sites are lost after the glycosylation is removed. Our laboratory is working on a project to improve methods of positively identifying both N- and O-linked sites. We have a specific interest in not only identifying the sites, but also in determining all of the glyco-structures on these peptides. Work is underway to develope the techniques for this approach, including the development of software to aid in the interpretation. Other software on the laboratory web site at http://sx102a.niddk.nih.gov continues to be in heavy demand. As an increased understanding of protein interactions becomes known, the importance of isolating protein complexes and identifying the players is of big interest. The group is interacting with several research groups working in this area and developing the techniques to deal with these samples. Of particular interest is the identification of all the proteins in a specific proteome. The common approach is to digest an entire proteome with trypsin and then fractionate this by two dimensional chromatography. This method ?discards? all the chromatographic information as the entire 2-D liquid chromatographic run must be analyzed as one search set. We have chosen to pre-separate the proteins in the proteome using reverse phase (C18) chromatography and separately digest and characterize each fraction, as this should give a more concise and accurate search. After using some trial protein mixtures, we chose to use the proteome of Rickettsia. Reckettsia is a class B biowarfare agent and thus very relevant to NIH following the new initiatives here. Protein samples were supplied from the University of South Alabama and involved just the soluble proteins. Of the 834 proteins in the proteome, 53 were identified in the initial trial run using fast chromatography and limited data acquisition. This number expanded a little when longer chromatographic runs were tried but the method is far from optimized. However this ?proteome? methodology is far from optimized and will be the subject of further development work. For full details of the results so far, view the web site at http://sx102a.niddk.nih.gov/proteome.html. Other projects include: (1) The characterization of cyclized Green Fluorescent Protein (GFP), which is also being used as a tag for proteomic approaches. Cyclase Associated Protein, mutant cyanovirin and dihydrofolate reductase have been effectively characterized. (2) The characterization of the molecular structure of silk proteins. This required the development of methods to determine isotopically enriched proteins. (3) A similar method has been established to identify and characterize abnormal amino acids found in natural products targeted for their anti-HIV and anti-cancer activity. (4) We are working on methods to elucidate structural information of HIV-integrase cross-linked with DNA and Actinomyces naeslundii fimbriae. (5) In order to identify the most reactive sites in tubulin, every cysteine in the tubulin has been reacted with various sulfhydryl reagents. This method will be a very important tool in protein-drug binding studies as well as for understanding tubulin polymerization mechanism. (6) A method to characterize fluxes of palmitoylation of the TRH receptor is under investigation. This research is important to understand how and why post-translational modifications are triggered by environmental changes. The laboratory runs a large number of more rountine analyses, helping investigators from all over NIH. The demand for characterization of proteins has increased so dramatically that a new LCMS obtained from elsewhere in the Institute has been set up in our group for hands-on use by those investigators. The QTOF instrument, which was jointly purchased by five institutes, runs on an almost continuous basis.